Passage through the animal cell cycle is regulated at least in part by the cyclin-dependent kinases, at least nine of which have been so far identified (Meyerson et al., EMBO J., 11:2909–2917, 1992; Pines, Semin. Cell Biol., 5:339–408,1994; Tassan et al., Proc. Natl. Acad. Sci. USA, 92:8871–8875, 1995; de Falco and Girdano, J. Cell. Physiol., 177:501–506, 1998). The functions of three of them, cdk1 (cdc2), cdk2 and cdk4, have been delineated in most detail. In brief, cdk1 plays a role in regulating the G2 to M-phase transition (Lohka et al., Proc. Natl. Acad. Sci. USA, 85:3009–3013, 1988; Draetta et al., Cell, 56:829–838, 1989; Labbe et al., EMBO J., 8:3053–3058, 1989; Nigg, Curr. Opin. Cell Biol., 5:187–193, 1993); cdk2 is involved in S-phase entry and progression (Dulic et al., Science, 257:1958–1961, 1992; Koffet al., Science, 257:1689–1693, 1992; Ohtsubo and Roberts, Science, 259:1908–1912, 1993; Sherr, Science, 274:1672–1677, 1996); and cdk4, by inactivating the growth-suppressing functions of the Rb-protein, appears to permit passage through G1 phase (Matsushima et al., Cell, 71:323–334, 1992; Kato et al., Genes & Develop., 7:331–342,1993; Sherr and Roberts, Genes & Develop., 9:1149–1163, 1995; Weinberg, Cell, 81:323–330, 1995). Perhaps the most direct demonstration for the roles of cdk1 and cdk2 was derived from experiments using dominant-negative forms of the proteins, which were mutated in their ATP-binding sites but could still form complexes with appropriate cyclins. Expression of the dominant-negative forms of cdc2 or cdk2 blocked cells in the G2 and G1 phases of the cycle, respectively (van den Heuvel and Harlow, Science, 262:2050–2054, 1993). Though it is less direct, other evidence supports a role for cdk4 in G1-phase progression through regulation of Rb function. For example, over-expression of D-type cyclins, which activate cdk4, shortens G1-phase length (Quelle et al., Genes & Develop., 7:1559–1571, 1993). It appears that certain cell lines which lack the Rb protein no longer require cyclin D-associated kinase activity for growth (Lukas et al., Molec. Cell. Biol., 15:2600–2611, 1995). Also, it was shown that microinjection of active cyclin D1-cdk4 complexes into quiescent human diploid fibroblasts induced a portion of the cells to enter S-phase (Connell-Crowley et al., Curr. Biol., 8:65–68, 1998).
More recently, the notion that cdk4 is essential for cell proliferation has been challenged by the finding that mice strains generated to be devoid of cdk4 are viable (Rane et al., Nature Genetics, 22:44–52, 1999; Tsutsui et al., Molec. Cell. Biol., 19:7011–7019, 1999). However, such mice are small in size, infertile and prone to the development of diabetes associated with a degeneration of pancreatic islets. In light of many previous in vitro studies, it was surprising to find that cdk4-deficient embryonic fibroblasts derived from such mice appeared to proliferate normally in culture. However, when such cells were growth-arrested in culture and then stimulated to re-enter the cell cycle, their entry into S-phase was markedly delayed when compared to wild-type, cdk4-containing cells (Rane et al., Nature Genetics, 22:44–52, 1999; Tsutsui et al., Molec. Cell. Biol., 19:7011–7019, 1999). Similar growth properties had in fact previously been observed in cells (derived from the U2OS line) having reduced cdk4 activity levels due to enforced expression of a dominant-negative form of cdk4 (Jiang et al., Molec. Cell. Biol., 18:5284–5290, 1998). Like the cdk4-negative embryonic fibroblasts, the cell line showed a reduced entry into S-phase after release from growth arrest, but appeared otherwise normal in growth properties. Taken together, the latter studies suggest that cdk4 may not be essential for cell growth and that its functions may be more important in some cell types than in others. Alternatively, it is possible that the normal functions of cdk4 have been at least partially assumed by another kinase, such as cdk6, in the absence of cdk4. The severity of the effects of cdk4 absence on certain organs or cell types may be in part a reflection of the normal balance of cdk4 and cdk6 in the particular cell types. Such a partial assumption of function is not unprecedented. A notable example is observed in Lck-deficient mice, in which a related Src-family kinase, Fyn, appears to partially substitute for Lck. Mice deficient in both Fyn and Lck unmask the essential roles of the two kinases in T cell development and signaling (Appleby et al., Cell, 70:751–763, 1992; Molina et al., Nature, 357:161–164, 1992; Groves et al., Cell, 5:417–428, 1996).
Among the cdk family, cdk4 and cdk6 form a sub-group extremely homologous in structure. Then their entire primary structures are compared, nearly 70% homology is observed (Meyerson et al., EMBO J., 11:2909–2917, 1992; Hanks, Proc. Natl Acad. Sci. USA, 84:388–392, 1987). Furthermore, the two proteins share numerous other similarities. Both are activated by binding to D-type cyclins (Bates et al., Oncogene, 9:71–79, 1994; Matsushime et al., Molec. Cell. Biol., 14:2066–2076, 1994; Meyerson and Harlow, Molec. Cell. Biol., 14:2077–2086, 1994; Lucas et al., J. Immunol., 154:6275–6284, 1995). Unlike cdk1 and cdk2, neither will phosphorylate the histone H1 protein in vitro, but will utilize the Rb protein as a substrate (Matsushime et al., Molec. Cell. Biol., 14:2066–2076, 1994; Meyerson and Harlow, Molec. Cell. Biol., 14:2077–2086, 1994). Unlike cdk1 and cdk2, cdk4 and cdk6 lack a regulatory threonine residue in their ATP binding sites (Meyerson et al., EMBO J., 11:2909–2917, 1992; Hanks, Proc. Natl. Acad. Sci. USA, 84:388–392, 1987; Morgan, Nature, 374:131–134, 1995). All animal cells yet examined contain both cdk4 and cdk6, but the ratio of the two proteins varies among cell types (Meyerson et al., EMBO J, 11:2909–2917, 1992). For example, T lymphocytes are an especially rich source of cdk6 activity (Meyerson and Harlow, Molec. Cell. Biol., 14:2077–2086, 1994; Lucas et al., J. Immunol., 154:6275–6284, 1995), while, as shown below, fibroblast cells, such as the 3T3 line, have very high levels of cdk4. Because of their many similarities in structure and activity, it has been presumed, prior to the present invention, that cdk4 and cdk6 perform very similar functions in cells, with either cdk4 or cdk6 predominating in certain cell types and tissues. That cdk6 likely plays role(s) in G1 phase, like cdk4, has been suggested by analysis of lines with excess or deficient cdk6 activities due to expression of wild-type or dominant-negative forms of the kinase (Grossel et al., J. Biol. Chem., 274:29960–29967, 1999; Ojala et al., Cancer Res., 59:4984–4989, 1999). Enforced expression of wild-type cdk6, either alone (Grossel et al., J. Biol. Chem., 274:29960–29967, 1999) or together with cyclin D1 (Ojala et al., Cancer Res., 59:4984–4989, 1999) appeared to shorten G1 phase length whereas the dominant-negative form delayed S-phase entry (Grossel et al., J. Biol. Chem., 274:29960–29967, 1999). These studies were both performed with the U2OS cell line, which is defective in production of the p16INK4a cdk inhibitor (Koh et al., Nature, 375:506–510, 1995; Lukas et al., Nature, 375:503–506, 1995; Medema et al., Proc. Natl. Acad. Sci. USA, 92:6289–6293, 1995). Furthermore, the level of enhancement or abrogation of cdk6 in the transfected U2OS cells was not demonstrated in those studies.
From examinations of cdk4 and cdk6 activities in T lymphocytes, similarities in the regulation of the two activities emerged. When normal resting T cells were stimulated to enter the cell cycle, cdk4 and cdk6 activities were the first to be detected (Meyerson and Harlow, Molec. Cell. Biol., 14:2077–2086, 1994; Lucas et al., J. Immunol., 154:6275–6284, 1995; Lucas et al., J. Cell. Physiol., 165:406–416, 1995). Each kinase formed two different complexes, with cyclin D2-associated activities preceding by many hours the appearance of cyclin D3-associated activities (Meyerson and Harlow, Molec. Cell. Biol., 14:2077–2086, 1994; Lucas et al., J. Immunol., 154:6275–6284, 1995). Increases in cdk4, cdk6 and cyclin D2 protein levels and in both cdk4 and cdk6 kinase activities occurred very early in G1-phase, before synthesis of IL-2, the major T-cell “progression” factor and thus before IL-2/IL-2 receptor interactions which are essential for progression through the restriction point and cell proliferation (Lucas et al., J. Immunol., 154:6275–6284, 1995; Lucas et al., J. Cell. Physiol., 165:406–416, 1995; Modiano et al., J. Biol. Chem. 269:32972–32978, 1994). However, differences in the properties of the two kinases also emerged. Thus, cdk6 protein was readily detected in resting T cells (Lucas et al., J. Immunol., 154:6275–6284, 1995). Increases in the levels of this protein and its kinase activity preceded by at least two hours those of cdk4 (Lucas et al., J. Immunol., 154:6275–6284, 1995; Lucas et al., J. Cell. Physiol., 165:406–416, 1995). It was further demonstrated by immunofluorescence microscopy that the resting population of T-cells contained cdk6 and that within minutes of T-cell activation it was translocated to the nucleus (Nagasawa et al., J. Immunol., 158:5146–5154, 1997), suggesting a very early role for the protein in cell cycle entry or early G1-phase progression. The T-cell derived Jurkat cell line, like its normal counterpart, contains a high level of cdk6 protein. Cdk6 kinase activity can be detected in high amount in the normal proliferative state of the cell line. However, when the line is stimulated by agents which stimulate IL-2 production, such as a combination of anti-CD3 and anti-CD28 monoclonal antibodies, there is a dramatic and rapid (within 15 minutes) increase in cdk6 kinase activity and a translocation of cdk6 and cyclin D2 proteins to the cell nuclei (Nagasawa et al., J. Immunol., 158:5146–5154, 1997). Taken together, the latter observations suggested that cdk6 is performing important functions very soon after cells are stimulated through receptors which initiate signal transduction pathways leading to cell growth and/or the production of growth factors involved in cell cycle progression.
It is therefore of interest to determine the effects of both cdk4 and cdk6 in various cell systems, especially since it appears likely that the functions of cdk4 and cdk6, and their relative importance, may differ in various cell types, as discussed further below.
Many different molecular defects have been observed in breast cancer cells. Although the outcome is malignant cell growth, no one defect or set of defects is seen in all breast tumors. However, as knowledge of signal transduction and cell cycle pathways has increased, it has become clear that these defective molecules are components of basic regulatory networks and that defects in any of a number of them can lead to the same or very similar phenotypes (Hunter, Cell, 88:333–346, 1997). Two key components of the fundamental cell-growth regulatory networks are the Rb and p53 proteins (Weinberg, Cell, 81:323–330,1995; Levine, Cell, 88:323–331,1997). Both are growth-suppressing molecules and defects in either protein or in proteins controlling their activities are seen in virtually all human tumors (Levine, Cell, 88:323–331, 1997; Hollstein et al., Nucleic Acids Res., 22:3551–3555, 1994). Rb activity is regulated in part by the G1-phase cyclin-dependent kinases, such as cdk4, which are in turn regulated by CDKIs (cyclin-dependent kinase inhibitors of the Cip/Kip and Ink4 families), cyclin D-family members, and cdc25 phosphatases (Sherr, Science, 274:1672–1677, 1996; Morgan, Nature, 374:131–134, 1995). Overproduction of cyclin D1 has been observed in a relatively high portion of human breast tumor samples (Buckley, et al., Oncogene, 8:2127–2133, 1993; Gillett et al., Cancer Res., 54:1812–1817, 1994; Sutherland et al., Acta Oncologica, 34:651–656, 1995; Weinstat-Saslow et al., Nature Medicine, 1:1257–1260,1995); decreased production of the CDKIs p16 (ink4a) orp27 (kip1) has been seen in others (Cairns et al., Nature Genetics, 11:210–212., 1995; Tan et al., Cancer Res., 57:1259–1263, 1997); and dysregulatien of cdc25A has been seen in some tumor cell lines (Galaktionov et al., Science, 269:1575–1577, 1995). In turn, p53 regulates production of p21 (cip1), also an inhibitor of cdks (E1-Diery et al., Cell, 75:817–825, 1993; Sherr and Roberts, Genes & Develop., 9:1149–1163, 1995), and Bax (Miyashita and Reed, Cell, 80:293–299, 1995), an inducer of programmed cell death. Production of mutant p53 has been seen in many breast tumors (Levine, Cell, 88:323–331, 1997; Hollstein et al., Nucleic Acids Res., 22:3551–3555, 1994; Bartek et al., Int. J. Cancer, 46:839–844, 1990); decreased Bax production has also been observed in some (Krajewski et al., Cancer Res., 55:4471–4478, 1995).
As noted above, cdk4 is a major regulator of Rb function (Sherr, Science, 274:1672–1677, 1996) and cellular alterations or mutations which interfere with its activity are likely candidates for oncogenic events. However, such defects may also interfere with cdk6 activity, which, as discussed above, is a kinase which is highly related to cdk4 in structure (Hanks, Proc. Natl. Acad. Sci. USA, 84:388–292,1987; Meyerson et al., EMBO J., 11:2909–2917, 1992) and regulated by the same basic set of molecules, that is Cip/Kip and Ink4-family CDKIs, D-type cyclins, and cdc25 phosphatases (Morgan, Nature, 374:131–134, 1995; Sherr and Roberts, Genes & Develop., 9:1149–1163, 1995). Amplification of cdk6 DNA and increased cdk6 enzyme levels have been seen in some human gliomas and squamous cell carcinoma lines, respectively (Costello et al., Cancer Res., 57:1250–1254, 1997; Timmermann et al., Cell Growth Different., 8:361–371,1997), but the possible clinical significance of these observations remains to be elucidated.
Therefore, there is a need to further elucidate the functions of cell cycle regulators such as cdk4 and cdk6 and to use such information to develop a better understanding of cell cycle events in tumor cells so that therapeutic strategies can be devised.